Aim: To study whole blood hemolysis with ammonium chloride solution.
Principle:
NH4Cl is the active agent in most RBC lysis buffers. NH3 diffuses freely through the cell membrane. Once
inside the cell, a new equilibrium between NH3 and NH+4 is established.
(1) H2O + NH3 <-> NH+4 + OH-
RBCs (and not leukocytes) have a membrane Cl-/ HCO-3 exchanger. With the Cl- in the lysis buffer
-
coming in, the HCO 3 is going to exit the cell. Inside the cell, there is the carbonic anhydrase catalyzed
equilibrium:
(2) CO2 + OH- <-> HCO-3
This equilibrium will be shifted to the right inside the cell as the HCO-3 is exchanged for Cl-. This
means OH is being consumed, which drives equilibrium (1) towards the right. The overall result is
net influx of NH4Cl into the RBCs which results in increased osmotic pressure until the cells burst
from the water influx.
Procedure:
Collect blood in EDTA vial.
Transfer the blood (500uL) in 50mL falcon and add 25mL RBC lysis buffer and incubate for 45min
at room temperature.
Then add 25mL PBS (1X) to it and centrifuge at 1000g (40 C) for 15 min.
Discard the supernatant and resuspend the pellet in 1 mL PBS.
Transfer it to 1mL tube and centrifuge at 1000g for 15 min.
Discard the supernatant and resuspend the cells in PBS and observe under the microscope.
Observations: Before hemolysis: blood smear stained with Leishman stain showed presence of RBCs
(pink colored enucleated cells).
After hemolysis: we observed white pellet of WBCs. Hemolysis was further confirmed by Leishman
staining of WBCs wherein we observed nucleated cells only.
Discussion:
A typical native mature erythrocyte has a biconcave discoid shape. In normal conditions, it maintains
its volume, ionic composition, pH and membrane structure and integrity. When an erythrocyte is
placed in a hemolytic medium, it becomes unstable, and its volume changes. If the hemolysis is of
colloid-osmotic type, the cell swells and eventually lyses when the volume exceeds a critical level.
The capacity of erythrocytes to resist hemolysis can be modified by various pathologies and by a wide
variety of physical, chemical or pharmacological treatments. Its study can thus yield relevant
information to hematologists and clinicians for diagnostic purposes or fundamental researches. A
widely used hemolysing agent is ammonia chloride in a proper buffer solution. The membrane is
impermeable to NH4+ and the hemolysis is due to diffusion of free NH3 across the membrane. Once
inside the cell, a new equilibrium between NH3 and NH4+ is established and the intracellular
OH− exchanges to the extracellular Cl− resulting in a net influx of NH4Cl. Importantly, the transport
of OH− is largely mediated by CO2/HCO3− exchange, so that inhibition of carbonic anhydrase or
depletion of CO2 slows the lysis. In the presence of CO2 the rate of hemolysis was observed to be
proportional to the rate of influx of the anion, provided that the NH3 permeability is not rate limiting.
Nowadays, it is established that the rate-limiting factor of the hemolysis in isotonic NH4Cl under
typical conditions (in the presence of HCO3−) is the Cl−/HCO3− transmembrane exchange through
Band 3 anion channel. Therefore, study of hemolysis in isotonic ammonium chloride solution may
lead to the evaluation of the amount of Band 3 exchangers on the erythrocyte membrane.
Blood collection in EDTA vial
Addition of lysis buffer to the blood
Blood smear of whole blood was prepared and stained with
Leishman stain. After staining, enucleated RBCs were observed under the microscope.
After RBC lysis, white pellet of WBCs was observed
RBC pellet observed in whole blood while white pellet of WBCs was obserbed after
hemolysis.
Leishman stained WBCs after hemolysis.
